Effect of diverse mammalian and nonmammalian gonadotropins on isolated rat Leydig cells

The isolated rat Leydig cell assay was employed to study species specificity in biological action. Eight mammalian luteinizing hormones (LHs), human chorionic gonadotropin, and pregnant mare serum gonadotropin stimulated testosterone production in this assay with a 250-fold potency range. A high potency correlated with a high carbohydrate content. Comparison of the potencies of these hormones in the Leydig assay, the ovarian ascorbic acid depletion assay, and the Xenopus ovulation assay showed the results of the two rat assays to be the most similar, demonstrating that the receptor as well as the hormone play a role in determining species specificity. The LH specificity of the assay was reexamined and follicle-stimulating hormone (FSH) was found to have a small intrinsic activity. Nonmammalian LHs, which generally possess little or no activity in in vivo mammalian assays, were able to stimulate rat Leydig cells, at 1000x mammalian LH doses. LH specificity was retained with nonmammalian LHs but the potency differences between nonmammalian LHs and FSHs were much less than mammalian LHs and FSHs.     

Modulation of steroidogenic activities in testis Leydig cells

Recent developments in the regulation of testicular Leydig-cell function support the following conclusions. (1) Enzymic activities involved in steroid production in the testis are mainly localized in Leydig cells. The aromatase enzyme complex for oestrogen production appears to be localized in Leydig cells as well as in Sertoli cells. (2) LH- (or hCG-) induced alterations of Leydig cells depend on dose and duration of exposure of the cell to the hormone. Locally produced oestradiol is probably involved in the inhibition of steroidogenesis. (3) The stimulatory action of LH on Leydig cells involves different proteins in concert with the activation of the cleavage activity of mitochondrial cholesterol side-chain. However, most of the functional properties of these proteins are yet unknown. (4) Different populations of Leydig cells are present in the testis. These different cell populations can be characterized by quantitatively and qualitatively different responses to hormones.     

Cyclin-dependent kinase 5 regulates steroidogenic acute regulatory protein and androgen production in mouse Leydig cells

The roles of cyclin-dependent kinase 5 (Cdk5) in central nervous system and neurodegenerative diseases have been intensely investigated in recent decades. Because protein expressions of Cdk5 and its regulator, p35, have been identified in Leydig cells, it is informative to further explore the novel function of Cdk5/p35 in male reproduction. Here we show that Cdk5/p35 protein expression and kinase activity in mouse Leydig cells are regulated by human chorionic gonadotrophin (hCG) in both dose- and time-dependent manners. Blocking of Cdk5 by molecular inhibitors or small interfering RNA resulted in reduction of testosterone production by Leydig cells. cAMP, a second messenger in LH signaling, was identified as a factor in hCG-dependent regulation of Cdk5/p35. Importantly, Cdk5 protein and kinase activity could support accumulation of steroidogenic acute regulatory (StAR) protein, a crucial component of steroidogenesis. We additionally addressed the protein interaction between Cdk5/p35 and StAR. The Cdk5-dependent serine phosphorylation of StAR indicated a possible mechanism by which Cdk5 induced accumulation of StAR protein. In conclusion, Cdk5 modulates hCG-induced androgen production in mouse Leydig cells, possibly through regulation of StAR protein levels. These results indicate that Cdk5 may play an important role in male reproductive endocrinology and is a potential therapeutic target in androgen-related diseases.     

Isolation and characterization of Leydig cells from adult bonnet monkeys (Macaca radiata): evidence for low steroidogenic capacity in monkey Leydig cells in contrast to rat Leydig cells

Most of the available information on Leydig cells has been obtained using a rodent model system. With an objective to extend the observations made with rat Leydig cells (RLCs) to primates, a method has been developed to isolate Leydig cells from monkey (Macaca radiata) testis. Enzymatic dissociation of monkey testis followed by Percoll-gradient fractionation of the interstitial cells resulted in the recovery of Leydig cells at densities corresponding to 1.064-1.070 g/ml. Purified (90-94%) monkey Leydig cells (MLCs) stained positive for the Leydig cell marker 3beta-hydroxysteroid dehydrogenase. The cells responded to in vitro addition of human chorionic gonadotropin (hCG) and produced testosterone. Comparison of the in vitro testosterone-producing ability of MLCs with RLCs revealed that MLCs have much less steroidogenic capacity compared with the RLCs. Analysis revealed that limitation in substrate availability to mitochondrial P(450) side chain cleavage enzyme and low mitochondrial and smooth endoplasmic reticulum content in MLCs could be the possible reasons for the low steroidogenic capacity of the MLCs.     

Hormone-induced homologous and heterologous desensitization in the rat adipocyte

We examined the process of desensitization in the isolated rat adipocyte. When adipocytes were exposed to isoproterenol (10(-7) or 10(-5) M) or ACTH (250 mU/ml) for 2 h, there was a marked decline of as much as 77% in response upon restimulation by hormone, as measured by glycerol release or cAMP levels. This desensitization was both heterologous as well as homologous. Thus, for example, exposure of adipocytes to isoproterenol desensitized them to further stimulation by both isoproterenol and ACTH. The process was time dependent, since augmentation rather than desensitization was seen if cells were initially exposed to hormone for 30 min rather than 2 h. No desensitization was seen when the cells were restimulated with the nonhormonal lipolytic agent dibutyryl cAP. Similarly, no desensitization was seen when cells were first exposed to dibutyryl cAMP and then restimulated with hormone. We draw the following conclusions. First, desensitization in the adipocyte is a time- and dose-dependent process that is specific for adenylate cyclase-activating hormones. Secondly, the process is heterologous as well as homologous. Initial exposure of the adipocyte to one adenylate cyclase-activating hormone reduces its adenylate cyclase or lipolytic response upon reexposure to either the same or a different adenylate cyclase-activating hormone. Finally, the reduction in the end result of hormone activation, lipolysis, is due in part to a decrease in inducible levels of cAMP.     

老年雄性大鼠睾丸类固醇合成酶与间质细胞形态的变化

目的通过测定老年雄性sD大鼠睾丸类固醇合成急性调节蛋白（StAR）、1713-羟类固醇脱氢酶（1713-HSD）表达，观察睾丸间质和kydig细胞结构的变化，探讨老年大鼠性腺功能减退的机制。方法用人绒毛膜促性腺激素（hC6）连续刺激3月龄和24月龄雄性SD大鼠，测定基础状态和hCG刺激后血清睾酮（T）、睾丸StAR、17β-HSD Ⅲ mRNA的表达，对不同年龄大鼠睾丸间质和Leydig细胞进行光镜和电镜检查。结果（1）老年SD大鼠基础状态和hCG刺激后血清T水平和17β-HSD Ⅲ mRNA的表达显著低于青年大鼠，StAR mRNA表达无明显变化；（2）老年大鼠睾丸间质中成纤维细胞增生明显而Leydig细胞数量减少，并出现退行性改变，hCG刺激8d后，青年和老年大鼠两组睾丸形态学均无明显变化。结论雄性大鼠睾丸17β-HSD Ⅲ mRNA表达以及teydig细胞形态学随着增龄的明显异常可能是老年SD大鼠T合成减少的主要影响因素。     

Differences in the control of sterol metabolism between mouse and rat Leydig cells

The metabolism of hydroxysterols, which bypass the cAMP-dependent, cycloheximide-inhibitable transport to cytochrome P-450 side-chain cleavage enzyme complex (P-450scc) required by cholesterol, and whose metabolism exceeds that of cholesterol in luteal cells, has been investigated in primary cultures of Leydig cells purified from the mouse and the rat. An unexpected finding was that metabolism of 25-hydroxycholesterol by mouse Leydig cells was far lower than cAMP-stimulated cholesterol metabolism. The metabolism of 20 alpha-hydroxycholesterol and 22R-hydroxycholesterol was equivalent to and greater than, respectively, maximal cholesterol metabolism by mouse Leydig cells. As expected, metabolism of 25-hydroxycholesterol by rat Leydig cells was much greater than cholesterol metabolism, as was metabolism of 20 alpha-hydroxycholesterol and 22R-hydroxycholesterol. Hydroxysterol metabolism was not increased by cAMP. Cycloheximide abolished the cAMP-stimulated increase in testosterone production by Leydig cells of both species but had no effect on metabolism of any of the hydroxysterols by Leydig cells of either species. In addition, it was shown that the relatively low rate of 25-hydroxycholesterol supported testosterone production in mouse Leydig cells was not due to inhibition of the conversion of pregnenolone to testosterone. It is concluded that a species-specific difference in the control of mitochondrial sterol metabolism exists between the rat and the mouse. The data suggest that either the P-450scc differs between mice and rats or that an effector of P-450scc, which greatly facilitates the binding and metabolism of cholesterol, is of particular importance in the control of sterol metabolism in the mouse Leydig cell.     

Carbachol induces homologous steroidogenic refractoriness of bovine fasciculata-reticularis cells

Recently it was shown that the cholinergic agonist carbachol stimulates cortisol production in bovine ZFR cells via muscarinic receptor M(3). In the present study, we investigated the effect of chronic cholinergic stimulation on steroidogenic response and muscarinic receptor regulation in ZFR cells. Cortisol secretion of ZFR cells treated with 10(-4) M of carbachol decreased in a dose- and time-dependent manner. The carbachol-elicited loss of response was associated with a decrease in M(3) receptor number, which was also time- and dose-dependent. The down-regulation of the receptors was not associated with the decrease of M(3) receptor mRNA level. The marked steroidogenic desensitization caused by pretreatment of carbachol did not alter ACTH or angiotensin II activated steroid response. Northern blot analysis showed that carbachol pretreatment did not change the gene expression of P450scc, P450cl7, 3betaHSD and StAR mRNAs. These results suggest that carbachol induces homologous steroidogenic refractoriness of ZFR cells.     

Induction of an estrogen-dependent early steroidogenic lesion in murine Leydig tumor cells

The effects of low doses (37 pM to 3.7 nM) of 17 beta-estradiol on Leydig tumor cell steroidogenesis were studied in primary culture. This gonadotropin-responsive Leydig tumor line (M5480A) produces progesterone as the major steroid and lower levels of testosterone. It was found that these tumor cells possess a relatively high level of estradiol receptors, but only low levels of estradiol. We, therefore, maintained dispersed Leydig tumor cells in culture under basal or hCG-stimulated conditions for varying periods of time with or without graded doses of estradiol. The media from these cultures were analyzed for pregnenolone, progesterone, and testosterone by specific RIAs. Although testosterone levels were similar to control values, both pregnenolone and progesterone levels were significantly decreased by low doses of estradiol in a dose- and time-dependent manner. For example, basal progesterone levels were diminished 36% by 0.37 nM estradiol, and this effect could be reversed by the antiestrogen LY117018 [6-hydroxy-2-(p-hydroxyphenol)benzo-b-thien-3-yl-p-2-(1-pyrr olidinyl)- ethoxyphenyl ketone]. To evaluate whether the decreased medium progesterone level was due to increased metabolism, [3H] progesterone was added to estrogen-treated and control cells, and ether-extracted media were analyzed for steroid metabolites by HPLC. No significant difference in progesterone metabolism, including its conversion to testosterone, was detected between control and treated cells. Thus, the estradiol-mediated decrease in progesterone concentrations most likely reflects decreased synthesis rather than increased metabolism. These results provide the first indication of an estrogen-mediated effect at an early site in Leydig tumor cell steroidogenesis.     

Expression of 11beta-hydroxylase in rat Leydig cells.

11Beta-hydroxy (11beta-OH) derivatives of certain steroids function as inhibitors of 11beta-hydroxysteroid dehydrogenase isoform 1 (11betaHSD1), an enzyme expressed in Leydig cells that catalyzes the reversible oxidation of biologically active glucocorticoids to inactive 11-dehydro metabolites. 11beta-Hydroxylase is an adrenal enzyme responsible for glucocorticoid biosynthesis, catalyzing 11beta-hydroxylation of steroids and thus producing 11beta-OH-steroid derivatives. The aims of the present study were 1) to examine whether 11beta-hydroxylase is expressed in testis, 2) to define the biochemical characteristics of the testicular form of this enzyme, and 3) to establish whether 11beta-hydroxylated steroids inhibit Leydig cell 11betaHSD1 activities. 11beta-Hydroxylase mRNA was detected in purified rat Leydig cells by RT-PCR. Sequencing confirmed that the PCR products had 100% identity with the published rat adrenal enzyme cDNA sequence. Immunohistochemistry and Western blot analysis using a mouse monoclonal antibody confirmed the expression of 11beta-hydroxylase protein in Leydig cells. Moreover, 11beta-hydroxylase activity, synthesis of corticosterone from 11-deoxycorticosterone, was measurable in Leydig cells, and the K(m) and maximum velocity values were 7.28 +/- 0. 92 microM and 1.13 +/- 0.04 micromol/10(6) cell x h, respectively. When assayed in Leydig cells, several 11beta-hydroxylated steroids were efficient inhibitors of 11betaHSD1 dehydrogenase activity, whereas other 11-keto compounds were effective as inhibitors of oxidoreductase activity. These results provide the first direct evidence that rat Leydig cells express 11beta-hydroxylase, which may be involved in the regulation of glucocorticoid metabolism within the testis through local biosynthesis of endogenous inhibitors of 11betaHSD1.     

Cholesterol synthesis in isolated rat hepatocytes: effect of homologous and heterologous serum lipoproteins.

The present study investigated the effect of serum lipoproteins on sterol synthesis by isolated rat hepatocytes. These cells were maintained in culture medium for 24 hr and incubated for the same period of time with increasing concentrations of serum lipoproteins (5-150 microgram of lipoprotein-protein per ml) isolated from different animal species. The viability of the cells was ascertained by their ability to synthesize cholesterol and protein and to secrete serum proteins into the medium. Rat VLDL and LDL did not alter sterol synthesis, which was stimulated instead by HDL. Rat serum chylomicrons were also ineffective. Human LDL significantly reduced the synthesis of sterols from both acetate and tritiated water; this effect was also induced by human VLDL to a reduced extent. VLDL isolated from hypercholesterolemic rabbit (VLDLC) strongly inhibited sterol synthesis from acetate but not from mevalonate. Cholesteryl-ester-rich VLDL isolated from a patient with type III hyperlipidemia (type III VLDL) were more effective than normal VLDL in suppressing sterol synthesis from acetate. The implications of these findings are discussed with regard to the possible role of cholesteryl-ester-rich lipoproteins on the in vivo regulation of sterol synthesis in the liver.     

Autocrine regulation of steroidogenic function of Leydig cells by transforming growth factor-alpha

We have determined the effects of LH on the expression of transforming growth factor-alpha (TGFalpha) and epidermal growth factor receptor (EGFR) system in rat Leydig cells and investigated its role in steroidogenesis. LH and TGFalpha/epidermal growth factor (EGF) significantly increased the levels of TGFalpha mRNA and protein, and the levels of EGFR protein in immature rat Leydig cells (ILC). Treatment with TGFalpha or EGF for 24h resulted in significant increase in androgen production in ILC. The increase in androgen production in response to TGFalpha was associated with increased mRNA levels of SR-BI, steroidogenic acute regulatory (StAR) and P450scc but not of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and P450c17. TGFalpha also caused a marked increase in the levels StAR protein in ILC. EGFR inhibitor (AG1478) blocked the effects of TGFalpha while MEK-inhibitor (PD98059) potentiated TGFalpha or LH effects on steroidogenesis. A PKA inhibitor (H89) blocked both TGFalpha and LH effects on steroidogenesis. We conclude that TGFalpha plays an autocrine role in LH dependent development and function of Leydig cells.